This invention relates to a pure crystalline form of a parathyroid hormone. More particularly, the invention relates to the crystalline form of teriparatide, PTH(1-34), and methods of preparation and purification of the fragmented hormone.
Parathyroid hormone (PTH) is a secreted, 84 amino acid product of the mammalian parathyroid gland that controls serum calcium levels through its action on various tissues, including bone. Studies in humans with certain forms of PTH have demonstrated an anabolic effect on bone, and have prompted significant interest in its use for the treatment of osteoporosis and related bone disorders.
Using the N-terminal 34 amino acids of the bovine and human hormone for example, which by all published accounts are deemed biologically equivalent to the full length hormone, it has been demonstrated in humans that parathyroid hormone enhances bone growth particularly when administered in pulsatile fashion by the sub-cutaneous and intravenous routes. A slightly different form of PTH, human PTH(1-38) has shown similar results.
PTH preparations have been reconstituted from fresh or lyophilized hormone, and incorporate various forms of carrier, excipient and vehicle. Most are prepared in water-based vehicles such as saline, or water acidified typically with acetic acid to solubilize the hormone. The majority of reported formulations also incorporate albumin as a stabilizer (see for example Reeve at al., Br. Med. J., 1980, 280:6228; Reeve at al., Lancet, 1976, 1:1035; Reeve at al., Calcif. Tissue Res., 1976, 21:469; Hodsman et al., Bone Miner; 1990, 9(2):137; Tsai et al., J. Clin. Endocrinol Metab., 1989, 69(5):1024; Isaac et al., Horm. Metab. Res., 1980, 12(9):487; Law et al., J. Clin Invest. 1983, 72(3):1106; and Hulter, J. Clin Hypertens, 1986, 2(4):360). Other reported formulations have incorporated an excipient such as mannitol, which is present either with the lyophilized hormone or in the reconstitution vehicle. Formulations representative of those employed for human studies include a human PTH(1-34) (SEQ ID NO: 2) preparation consisting, upon reconstitution, of mannitol, heat inactivated human serum albumin, and caproic acid (a protease inhibitor) as absorption enhancer (see Reeve at al., 1976, Calcif. Tissue Res., 21, Suppl., 469-477); a human PTH(1-38) preparation reconstituted into a saline vehicle (see Hodsman et al., 1991, 14(1), 67-83); and a bovine PTH(1-34) preparation in aqueous vehicle pH adjusted with acetic acid and containing albumin. There is also an International Reference preparation which for human PTH (1-84) (SEQ ID NO: 1) consists of 100 ng of hormone ampouled with 250 xcexcg human serum albumin and 1.25 mg lactose (1981), and for bovine PTH (1-84) consists of 10 xcexcg lyophilized hormone in 0.01 M acetic acid and 0.1% w/v mannitol (see Martindale, The Extra Pharmacoepia, The Pharmaceutical Press, London, 29th Edition, 1989 at p. 1338).
Commercial exploitation of parathyroid hormone requires the development of a formulation that is acceptable in terms of storage stability and ease of preparation. Because it is a protein and thus far more labile than the traditionally small molecular weight drugs, however, the formulation of parathyroid hormone presents challenges not commonly encountered by the pharmaceutical industry. Furthermore, like other proteins that have been formulated successfully, PTH is particularly sensitive to oxidation, deamidation and hydrolysis and further requires that its N-terminal and C-terminal sequences remain intact in order to preserve bioactivity.
The present invention provides methods of preparing crystalline forms of a fragmented parathyroid hormone (PTH) which heretofore have not been reported. The advantages of a crystalline form for the hormone are purity of the product and storage stability. Thus, for example, a crystalline form of PTH may be easily dissolved in a sterile solution in vials for parenteral administration. As a crystalline material, PTH may also be formulated, if desired, into other compositions such as, for example, tablets, capsules or suppositories.
Accordingly, in a first aspect the present invention is a novel crystalline form of a parathyroid hormone selected from the group consisting of PTH(1-34). PTH(1-37), PTH(1-38) and PTH(1-41), and, in particular crystalline human PTH(1-34) (SEQ ID NO: 2), generically known as teriparatide in the form of tetragonal plates or cubic crystals, both having a space group P422 and the following unit cell constants: a=b=91.071 xc3x85, c=37.665 xc3x85, xcex1=xcex2=xcex3=90xc2x0.
A second aspect of the present invention is a process for purifying a parathyroid hormone including the steps of:
(a) providing an aqueous solution of said hormone at a concentration of about 5 to 40 mg per ml;
(b) mixing said solution with a reservoir solution comprising organic solvent at a concentration of about 5 to about 50 volume percent and a buffer at a concentration to maintain the pH between about 6.0 and about 12.0; and
(c) allowing the resulting solution to stand at a predetermined time at a temperature between about room temperature and about 4xc2x0 C. until plate-like crystals form.
A third aspect of the present invention is a process for purifying a parathyroid hormone where the steps include:
(a) providing an aqueous solution of said hormone at a concentration of about 5 to 40 mg/ml;
(b) mixing said solution with a reservoir solution comprising a phosphate or formate salt precipitant at the concentration of about 0.5M to 2.5M and, optionally, an aqueous buffer solution, or a mixture thereof, at a concentration to maintain a pH between about 4.0 and 6.0; and
(c) allowing the resulting solution to stand at a predetermined time at a temperature between about room temperature and about 4xc2x0 C. until cubic crystals form.
A fourth aspect of the present invention is a novel crystalline form of a parathyroid hormone selected from the group consisting of PTH(1-34), PTH(1-37), PTH(1-38), and PTH(1-41), and, in particular crystalline human PTH(1-34) (SEQ ID NO: 2) in the form of hexagonal crystals having a space group P622 and the following cell constants: a=b=30.169 xc3x85, c=110.597 xc3x85, xcex1=xcex290xc2x0, xcex3=120xc2x0.
Finally, as a fifth aspect, the present invention is a process for purifying a parathyroid hormone comprising:
(a) providing a solution of a parathyroid hormone selected from the group consisting of PTH(1-34), PTH(1-37), PTH(1-38), and PTH(1-41) at a concentration of about 5 to about 40 mg/ml in about 10 to about 30 volume percent glycerol;
(b) mixing said solution with a reservoir solution comprising organic solvent at a concentration of about 5 to about 50 volume percent, ammonium sulfate at a concentration of about 0.5M to 3.0M, and a buffer at a concentration to maintain the pH of the solution between about 3.0 and about 7.0; and
(c) allowing the resulting solution to stand at a predetermined time at a temperature between about room temperature and about 4xc2x0 C. until hexagonal crystals form.